Process for preparing carbamyl derivatives of α-hydroxy acids and the corresponding α-hydroxy acids

ABSTRACT

This invention relates to a process for preparing carbamyl derivatives of α-hydroxy acids by the hydrolysis of 5-substituted 2,4-oxazolidinediones. 
     The carbamyl derivatives can be further hydrolyzed to α-hydroxy acids. 
     The invention also relates to a process for the enzymatic hydrolysis of racemic 5-substituted 2,4-oxazolidinediones to give only one of the two possible optical isomers, i.e. the D-carbamyl-α-hydroxy acid. The free D-α-hydroxy acid can be obtained from the optically active carbamyl derivative by simple hydrolysis. Of particular interest is the case in which the D-α-hydroxy acid is D(-)mandelic acid. The enzymatic activity required for preparing the carbamyl derivative of D(-)mandelic acid has been found both in homogenized veal liver and in a series of microorganisms, including Agrobacterium radiobacter, Bacillus brevis, Bacillus stearothermophilus, Pseudomonas sp., Pseudomonas desmolytica, Pseudomonas fluorescens, Pseudomonas putida.

This invention relates to a process for preparing α-hydroxy acids by thehydrolysis of compounds of general formula (I): ##STR1## where R can bea substituted or unsubstituted aromatic or aliphatic residue.

According to the present invention, the compounds of general formula(I), i.e. 5-substituted 2,4-oxazolidinediones, can undergo a hydrolysisreaction which opens the ring in accordance with the following scheme:##STR2## The compound of general formula (II) obtained by hydrolysis isthe carbamyl derivative of a α-hydroxy acid, from which the α-hydroxyacid can be obtained by subjecting compound (II) to further hydrolysisin accordance with the scheme: ##STR3##

The final product (III) is the α-hydroxy acid. A further importantsubject matter of the present invention is a process for the directproduction of D-carbamyl-α-hydroxy acids by the steroselective enzymatichydrolysis of racemic mixtures of compounds of general formula (I). Inthis respect, it has been surprisingly found possible to enzymaticallyhydrolyse 5-substituted DL-2,4-oxazolidinediones in such a manner as togive only the D-carbamyl-α-hydroxy acid, i.e. only one of the twopossible optical isomers. The free α-hydroxy acid of D configuration canbe obtained from the optically active carbamyl derivative by simplehydrolysis. Compounds such as those described in formula (I) can beeasily prepared from the corresponding α-hydroxy acids by reacting themwith urea as described by Helge Aspelund in Acta Acad. Aboensins Math.and Phys. 22 (7) 12 (1961). Resolution of the racemic mixture iseffected by stereoselective hydrolysis of the oxazolidine ring carriedout by enzymes easily obtainable from cultures of various microorganismsor from extracts of animal organs such as veal liver. One particularcase, which is extremely interesting but does not limit the generalvalidity of the aforesaid process, is the preparation of D (-) mandelicacid ##STR4## which is an important intermediate in the preparation ofsemisynthetic antibiotics.

This intermediate is generally prepared by resolution of the racemicmixture by means of the formation of diastereo-isomer salts withoptically active natural bases such as brucine. These processes in anycase give a maximum theoretical yield of 50% in that the L enantiomermust be racemised before being recycled.

According to the present invention, the carbamyl derivative of D(-)mandelic acid can be advantageously prepared by the stereoselectiveenzymatic hydrolysis of the corresponding 5-phenyl-2,4-oxazolidinedione.A further considerable advantage of the process according to the presentinvention is that the enzymatic reaction substrate racemisesspontaneously under the hydrolysis conditions, so that at the end of thereaction the carbamate of the D-mandelic acid is obtained in astoichiometric quantity with respect to the starting substrate.

The reaction scheme is as follows: ##STR5##

D(-) mandelic acid is then obtained from the D-mandelic acid carbamateby hydrolysis in an acid environment.

The enzymatic activity required for preparing the D(-) mandelic acidcarbamate has been found both in homogenised veal liver and in thefollowing microorganisms: Pseudomonas, Archromobacter, Corynebacterium,Brevibacterium, Microbacterium, Arthrobacter, Agrobacterium, Aerobacter,Klebsiella, Serratia, Proteus, Bacillus, Micrococcus, Sarcina,Protaminobacter, Streptomyces, Actinomyces, Candida, Rhodotorula, Pichiaand Paecilomyces.

Microorganisms of the following kinds have proved particularly suitable:

Agrobacterium radiobacter NRRL B 11291

Bacillus Brevis NRRL B 11080

Bacillus stearothermophilus NRRL B 11079

Pseudomonas sp CBS 145.75

Pseudomonas sp CBS 146.75

Pseudomonas sp ATCC 11299

Pseudomonas desmolytica NCIB8859

Pseudomonas fluorescens ATCC 11250

Pseudomonas putida ATCC 12633

In carrying out the process according to the present invention, themicroorganisms of the aforesaid types are cultivated under aerobicconditions in culture media containing sources of nitrogen, carbon,phosphorus and mineral salts at a temperature of between 20° C. and 80°C. for a time of between 10 and 72 hours and at a pH of between 6.0 and8.0.

Glucose, lactate, acetate, corn steep liquor and lactose can be used assources of carbon.

Hydrolysed meat, casein or soya, ammonium salts, urea, hydrantoin etc.can be used as sources of nitrogen.

A suitable culture medium has, for example, the following composition:

    ______________________________________                                        Meat peptone          5      g                                                Meat extract          5      g                                                Glucose               5      g                                                Distilled water       1000   cc                                               pH                    7.0 ÷ 7.2                                           ______________________________________                                    

The D(-) carbamate of mandelic acid can be produced directly in thefermentation media containing the corresponding DL-2,4-oxazolidinedione,or can be produced by directly using the microbic paste as resting cellsor by using extracts thereof. The enzymatic complexes of the presentinvention are extracted from the bacterial paste by the normal methodsused in enzymology.

For this purpose the cells are disintegrated using suitable apparatussuch as the French Pressure-Cell Press, Manton Gaulin Homogenizer,rotatory disintegrators etc., or using ultrasonics.

Hydrolysis of the 5-substituted D,L-2,4-oxazolidinedione can be carriedout by adding the enzyme in the following forms to the reaction mixture:fresh cells, lyophilised cells, toluenised cells, acetonic powder orcrude or purified extracts. A further technical and economicalimprovement can be made by immobilising the enzymes by way ofcombination with macromolecular compounds by forming chemical bonds withthe matrix or ionic bonds, or by physical immobilisation.

The examples given hereinafter describe other methods of effecting thepresent invention, but are not limitative thereof.

EXAMPLE 1

A culture broth was prepared having the following combination:

    ______________________________________                                        Meat peptone          5      g                                                Meat extract          3      g                                                Glucose               5      g                                                Distilled water       1000   cc                                               ______________________________________                                    

The pH was adjusted to 7.2 with soda, and the medium was distributedinto 500 ml flasks in portions of 100 ml. After sterilising for 30minutes at 110° C., the flasks were innoculated with a culture of thePseudomonas CBS 145.75 slant strain containing the same medium with 2%of agar (DIFCO) and incubated for 24 hours at 30° C. under orbitalstirring (220 r.p.m.).

1 ml of this preculture (D.O. at 550 nm=0.250 dil×1:10) was placed infive 500 ml flasks containing 100 ml of the same medium, and the culturewas incubated at 30° C. under orbital stirring (220 r.p.m.) for 24 hours(D.O. at 550 nm=0.450 dil 1:10).

The cells were then collected, washed in physiological solution andfinally suspended in 100 ml of 0.1 M pyrophosphate buffer of pH 8.7containing 2 g of DL 5-phenyl-2,4-oxazolidinedione at a temperature of50° C.

After 70 hours of incubation under these conditions, hydrolysis to theD-mandelic acid carbamate was completed, as proved by polarimetricanalysis of the reaction mixture.

The carbamate was isolated from the reaction mixture after removing thecellular paste by centrifuging, then cooled and the pH adjusted to 2.5with concentrated HCl. The precipitate so obtained was filtered, washedwith cold H₂ O and dried under vacuum. 1.8 g of carbamate were obtained,its identity being proved by I.R. and NMR spectra and by elementaryanalysis.

The specific optical rotatory power, [α]_(D) ²⁵, of the alcoholicsolution of the carbamate obtained as heretofore described was -141.

Its melting point (with decomposition) was 169° C.

1.4 grams of carbamate were suspended in 100 ml of H₂ O, then heatedunder reflux for 4 hours. The aqueous solution thus obtained wasacidified and then extracted with ethyl ether, and the organic phase wasconcentrated under vacuum to dryness. 1.10 grams of crude mandelic acidwere obtained having a [α]_(D) ²⁵ of -120 (optical yield 76%).

When crystallised from water, the crude product had a M.P. of 130° C.and a [α]_(D) ²⁵ of -154.5 in water, against a M.P. of 133° C. and a[α]_(D) ²⁵ of -158 as described in the literature for D(-) mandelicacid.

EXAMPLE 2

1 g of acetonic powder of homogenised veal liver was added to a solutioncontaining 500 mg of DL-5-phenyl-2,4-oxazolidinedione in 50 ml of 0.1 Mpyrophosphate buffer having a pH of 8.5.

The reaction mixture so obtained was incubated at 30° C. for 40 hours.

The D mandelic acid carbamate was then recovered as described in example1.

380 mg of crude carbamate were obtained having a specific opticalrotatory power, [α]_(D) ²⁵, of -136 is ethanol. Acid hydrolysis of thecrude carbamate was then carried out, and the mandelic acid wasextracted as described in example 1, to give 300 mg of crude D mandelicacid having a [α]_(D) ²⁵ of -116 (optical yield 73.5%).

We claim:
 1. A process for preparing D-carbamyl-α-hydroxy acids of theformula ##STR6## consisting of subjecting 5-substitutedDL-2,4-oxazolidinediones of the general formula: ##STR7## where R is asubstituted or unsubstituted aromatic or aliphatic residue, tostereoselective enzymatic hydrolysis in the presence of an agentproduced from microorganisms of the following types:(a) Agrobacteriumradiobacter NRRL B 11291, (b) Bacillus brevis NRRL B 11080, (c) Bacillusstearothermophilus NRRL B 11079, (d) Pseudomonas sp CBS 145.75, (e)Pseudomonas sp CBS 146.75, (f) Pseudomonas sp ATCC 11299, (g)Pseudomonas desmolytica NC1B8859, (h) Pseudomonas fluorescens ATCC11250, and (i) Pseudomonas putida ATCC
 12633. 2. A process for preparingD-carbamyl-α-hydroxy acids as claimed in claim 1, wherein the agentshaving enzymatic activity in the stereoselective hydrolysis of5-substituted 2,4-oxazolidinediones are obtained from homogenised vealliver.
 3. A process for preparing D-carbamyl-α-hydroxy acids as claimedin claim 1, wherein the microorganisms are cultivated under aerobicconditions at a temperature of 20° C. and 80° C.
 4. A process forpreparing D-carbamyl-α-hydroxy acids as claimed in claim 1, wherein themicroorganism cultivation time is between 10 and 72 hours.
 5. A processfor preparing D-carbamyl-α-hydroxy acids as claimed in claim 1, whereinthe pH of the microorganism culture medium lies within the range 6-8. 6.A process for preparing D-carbamyl-α-hydroxy acids as claimed in claim1, wherein the D-α-hydroxy acid is D(-) mandelic acid, and the substrateof general formula (I) is DL-5-phenyl-2,4-oxazolidinedione.